Heart specimens were obtained immediately after sacrifice and fixed in 10% formalin for 48 h. Thereafter, the specimens were dehydrated and embedded in paraffin and then cut transversely into 5-μm sections. For immunohistochemistry, the sections were blocked with 10% BSA and incubated with primary antibodies against CD68 (1:100, Abcam, United States) overnight at 4°C. Then, the sections were incubated with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) and DAB (Gene Tech, Shanghai, China). For immunofluorescence, the sections were incubated with primary antibodies against CD68 (1:200, Abcam, United States), CD206 (1:200, R&D Systems, United States) and CD80 (1:200, R&D Systems, United States) overnight at 4°C. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was conducted using commercially available apoptosis detection kits (Roche, Basel, Switzerland). All images were obtained with a light or fluorescence microscope and analyzed using Image-Pro Plus6.0 software.
Anti rabbit hrp reagent
Manufactured by Gene Tech
Sourced in China
The Anti-rabbit HRP reagent is a laboratory tool used to detect and quantify the presence of rabbit-derived proteins or antigens in biological samples. The reagent contains Horseradish Peroxidase (HRP), an enzyme that catalyzes a colorimetric reaction, allowing for the visualization and measurement of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Apoptosis detection kit, by Roche (1 mentions)
Cd206, by R&D Systems (1 mentions)
3 3 diaminobenzidine (dab), by Gene Tech (1 mentions)
Apoptosis detection kit, by Merck Group (1 mentions)
Anti tnfa, by Abcam (1 mentions)
Anti 4 hydroxynonenal 4 hne, by Abcam (1 mentions)
Anti cd68, by Abcam (1 mentions)
Diaminobenzidine dab, by Gene Tech (1 mentions)
Photo imaging system h550l, by Nikon (1 mentions)
Dab kit, by Gene Tech (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
5 protocols using anti rabbit hrp reagent
1
Characterizing Cardiac Macrophage Phenotypes
2
Cardiac Tissue Analysis: Staining and Quantification
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Hematoxylin–eosin (H&E) and PSR staining was performed according to our previous description (Wu et al., 2015 (link)). The quantitative digital analysis system (NIH Image 1.6, National Institutes of Health, United States) was used to analyzed the infarct size, which was expressed as a percentage of the total LV area. The software, Image-Pro Plus 6.0, was used to analyzed interstitial collagen deposition by PSR staining. Immunofluorescence staining of Wheat Hemagglutinin (1:100, Invitrogen, United States) was used to detect cardiomyocytes size as previous study described (Santos-Gallego et al., 2016 (link)).
For immunohistochemistry staining, hearts were incubated with anti-CD68 (1:100), anti-TNFa (1:100), or anti-4-hydroxynonenal (4-HNE, 1:100) (Abcam, Cambridge, MA, United States) followed by incubation with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) and a peroxide-based substrate DAB kit (Gene Tech, Shanghai, China). Light microscopy (H550L, Tokyo, Japan) was used for analysis.
Apoptosis Detection Kit (Millipore, Temecula, CA, United States) was used to detect apoptosis according to the manufacturer’s instructions. Briefly, hearts were incubated with HRP-labeled dUTP followed by a peroxide-based substrate DAB kit. Microscopy (BX51, Olympus, Japan) was used to analyzed the apoptosis-positive cells.
For immunohistochemistry staining, hearts were incubated with anti-CD68 (1:100), anti-TNFa (1:100), or anti-4-hydroxynonenal (4-HNE, 1:100) (Abcam, Cambridge, MA, United States) followed by incubation with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) and a peroxide-based substrate DAB kit (Gene Tech, Shanghai, China). Light microscopy (H550L, Tokyo, Japan) was used for analysis.
Apoptosis Detection Kit (Millipore, Temecula, CA, United States) was used to detect apoptosis according to the manufacturer’s instructions. Briefly, hearts were incubated with HRP-labeled dUTP followed by a peroxide-based substrate DAB kit. Microscopy (BX51, Olympus, Japan) was used to analyzed the apoptosis-positive cells.
3
Immunohistochemistry and Immunofluorescence of Hearts
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For immunohistochemistry, paraffin-embedded hearts were cut transversely into 4- to 5-μm sections. Next, the sections were deparaffinized and blocked with 10% bovine serum albumin. Then, the sections incubated overnight at 4 °C with primary antibodies and anti-rabbit HRP reagent (Gene Tech, Shanghai, China) at 37 °C for 60 min. Finally, the sections were visualized with diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 2 min at 37 °C and mounted with neutral gums. The sections were examined under a light microscope (Nikon H550L, Tokyo, Japan). For immunofluorescence, the sections were autoclaved for antigen retrieval and then blocked with 10% goat serum for 10 min. Next, the sections were incubated with primary antibodies against CD206 overnight at 4 °C. The sections were rinsed with PBS for 20 min before incubating with two different IRDye® 800CW-conjugated secondary antibodies for 60 min and subsequently counterstained with the SlowFade Gold antifade reagent containing DAPI.
4
Evaluating Cardiac Tissue Morphology
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The harvested heart was fixed in 4% paraformaldehyde for 24 h, subsequently dehydrated in ethanol and xylene series, and finally embedded in paraffin. To evaluate the cross-sectional sizes of cardiomyocytes and cardiac interstitial fibrosis, 5-μm cardiac tissue slides were stained with hematoxylin and eosin (H&E) and picrosirius red (PSR). Subsequently, images of the slides were obtained by optical microscopy with a Nikon Photo-Imaging System (H550L, Tokyo, Japan). For cardiomyocytes sizes and interstitial fibrosis quantification, images were analyzed with the Image-pro Plus 6.0 software (Maryland, USA).
For immunohistochemistry of myocardial sFRP2 expression, the cardiac tissue sections were deparaffinized, blocked with 8% goat serum, and then incubated with 1:100 diluted anti-sFRP2 antibody overnight at 4 °C. After washing, the slides were incubated for 1 h with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) at 37 °C, rinsed, and developed with a peroxide-based substrate DAB kit for 5 min (Gene Tech, Shanghai, China). Finally, images of the slides were obtained by light microscopy with a Nikon Photo-Imaging System (H550L, Tokyo, Japan).
For immunohistochemistry of myocardial sFRP2 expression, the cardiac tissue sections were deparaffinized, blocked with 8% goat serum, and then incubated with 1:100 diluted anti-sFRP2 antibody overnight at 4 °C. After washing, the slides were incubated for 1 h with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) at 37 °C, rinsed, and developed with a peroxide-based substrate DAB kit for 5 min (Gene Tech, Shanghai, China). Finally, images of the slides were obtained by light microscopy with a Nikon Photo-Imaging System (H550L, Tokyo, Japan).
5
Quantifying Myocardial Injury and Inflammation
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Hearts were arrested in 10% KCl solution immediately after being obtained. After fixation with 10% formalin for 48 h, the heart specimens were embedded in paraffin and then sliced into 5-μm sections. The heart injury score and myocardial collagen volume were analyzed by hematoxylin and eosin (HE) staining and masson's trichrome staining, respectively. Moreover, these sections were also subjected to immunofluorescence staining. In brief, the sections were incubated with primary antibodies against CD14 (R&D Systems, USA), against CD16 (R&D Systems, USA) and against p-p65 (Abcam, United Kingdom) overnight at 4°C. Then, the sections were incubated with secondary antibodies [anti-rabbit HRP reagent (Gene Tech, Shanghai, China)] and 40,6-diamidino-2-phenylindole [DAPI (Gene Tech, Shanghai, China)]. All the figures were captured with fluorescence microscope, and Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, United States) was used for relative quantification.
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